Published on 13 August 2024
The study “Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers” explores the dynamic behavior of ferritin at the single-molecule level. Using optical Nanotweezers, researchers observed how ferritin’s structure responds to environmental changes like pH variations and ascorbic acid concentrations. Key findings include increased ferritin dynamics with ascorbic acid, leading to ferric core dissolution, and ferritin disassembly pathway at pH 2, revealing intermediate fragments during the process. This research advances understanding of ferritin’s role in iron metabolism and potential medical applications.
Ferritin, a spherical protein shell composed of 24 subunits, serves as an efficient iron storage and release system through its channels. Investigating the structure change of ferritin in response to various chemicals is essential for understanding the origins of iron-related diseases in humans and other organisms. Currently, our understanding of ferritin is mostly based on ensemble measurements, which only provide an average response of the protein population. The influence of chemicals on ferritin’s dynamics and iron release is barely explored at the single-protein level.
This study employs advanced optical nanotweezers and microfluidics, a label-free, single-molecule technique, to observe the flexibility and dynamics of individual ferritin molecules in real time. The goal is to monitor the conformation changes of these molecules in response to environmental factors such as pH variations and ligand concentrations.
This work utilizes optical nanotweezers to track and analyze the dynamic behavior of single ferritin molecules. As shown in Figure 1, an 852 nm laser focuses on a gold double-nanohole (DNH) structure using a 60× air objective with a numerical aperture (NA) of 0.85. The DNH is positioned in a customized flow cell with a channel height of 50 µm.
The DNH structure introduces a tightly confined optical field, exerting an optical gradient force to retain a single protein within the nanohole gap for an extended period (right inset of Figure 2). The intensity of the transmitted light through DNH is detected by a silicon avalanche photodiode (APD) and converted into a voltage signal. Due to the higher refractive index of the protein compared to water, trapping a protein introduces an increase in transmission, an effect known as dielectric loading (Fig. 2).
The microfluidic system consists in,
After trapping a protein, buffers with varying ascorbic acid concentrations or different pH levels are sequentially introduced into the chamber at a flow rate of 0.005 mL/min.
The research provides a detailed understanding of ferritin’s structural flexibility and disassembly kinetics at the single-molecule level. Key findings include:
Various reductants, like Ascorbic Acid (AA), help mobilize iron from the ferritin shell through specific channels. Patients with hemochromatosis, who have significant iron overload, often need ascorbate supplementation during chelation therapy due to lower ascorbate levels. However, high levels of ascorbate can damage proteins, lipids, and DNA by generating radicals through the Fenton reaction. In that context, understanding AA influence on ferritin conformation is essential to better understand those pathologies.
The increase concentration of AA accelerates the reduction of Fe³⁺ to Fe²⁺, leading to frequent movements of 3-fold channels on ferritin. This channel activities increase the flexibility of ferritin molecules, resulting in an increased noise in the optical signal, as shown in Figure 3. This noise, quantitated as root mean square (RMS), increases with the AA concentration and reaches to the highest at 5 mM AA (right inset of Figure 3).
In addition, high ascorbate concentrations can create an acidic environment, affecting ferritin dynamics and iron release, as ferritin disassembles at very low pH levels. This conformation modification is however very dynamic as it is also known that disassembled ferritin can reassemble at higher pH due to electrostatic interactions.
In this context, ferritin disassembly was observed at pH 2, indicated by a stepwise decrease in the transmission signal. The optical nanotweezer technique tracks the disassembly kinetics of single ferritin molecules, with the transmission signal correlating linearly with the volume of the trapped protein. Initially, the native ferritin, composed of 24 subunits, exhibits a high transmission level. Upon exposure to pH 2, protonation of hydrogen bonds between subunits causes the protein to disassemble, resulting in low transmission levels corresponding to the fragment size. The disassembly process in Figure 4 revealed four critical intermediate fragments: 22-mer, 12-mer, tetramer and dimer.
This study demonstrates the capability of optical nanotweezers and microfluidics to resolve the structural flexibility and disassembly pathway of single ferritin molecules. Understanding the conformational changes of ferritin in response to various chemicals is crucial for elucidating their roles in iron metabolism, thus facilitating the development of medical treatments for iron-related diseases. The single-molecule insights gained here may also inspire further engineering of ferritin structures, including the design of molecular machines and drug delivery platforms. The research highlights the exciting potential of this innovative approach to study ferritin and its interactions at an unprecedented level of detail. Enabled by the precision and control of Elveflow’s advanced microfluidic instruments, this work underscores Elveflow’s role as a leading partner in scientific discovery, empowering researchers to push the boundaries of molecular research and deepen our understanding of microfluidic applications.
Arman Yousefi is a final-year PhD student in the Advanced Optics and Photonics (AOP) group at Nottingham Trent University. After earning a master’s degree in materials science and characterization from the University of Tehran, Arman focused on magnetic hydrogel nanocomposites, resulting in several international publications. In 2021, Arman joined the AOP group at NTU under the supervision of Professor Mohsen Rahmani and Dr Cuifeng Ying, concentrating on nanophotonic structures for single-molecule sensing. Notably, their research involves monitoring single ferritin with optical nanotweezers, resulting in publications in high-quality journals such as Nano Letters and ACS Nano. Additionally, these findings were presented at international conferences such as Photon2022 in Nottingham, UK, and Single-Molecule and NanoSystems 2023 in Barcelona, Spain. Arman’s research interests include the nanofabrication of optical structures and devices that advance single-molecule detection techniques.
Dr Cuifeng Ying is Senior Lecturer at Nottingham Trent University, and the Theme Leader in the Advanced Optics and Photonics (AOP) group. She received her Ph.D. in Physics in 2013 from Nankai University under the supervision of Prof. Jian-Guo Tian, focusing on enhancing fluorescent signals for biosensing applications using photonic crystals and plasmonic nanocavities. Following her doctoral studies, she undertook a postdoctoral appointment with Prof. Yongsheng Chen at Nankai University, where she began single-molecule sensing using nanopores. In 2016, she joined the Biophysics group of Prof. Michael Mayer as a postdoctoral researcher at the Adolphe Merkle Institute, University of Fribourg.
Dr. Ying is an active biophysics researcher interested in utilizing engineered nanostructures for characterizing disease-related proteins at the single-molecule level. Her research focuses on using plasmonic nanostructures and synthetic nanopores to study the conformational dynamics of individual proteins. Cuifeng has developed widely accessible nanopore fabrication techniques, surface functionalization methods and data analysis algorithms to allow fingerprinting of single, unmodified proteins using solid-state nanopores. Additionally, she has utilized optical nanotweezers to manipulate and monitor the conformational dynamics and assembly/disassembly pathways of single proteins in solution.
Yousefi A, Zheng Z, Zargarbashi S, Assadipapari M, Hickman GJ, Parmenter CD, Bueno-Alejo CJ, Sanderson G, Craske D, Xu L, Perry CC. Rahmani M, and Ying C, Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers. ACS Nano 18, 24, 15617–15626 (2024).
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